Faculty Bibliography

We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits, respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50 = 200 microM) and by aluminum lactate (IC50 = 400 microM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 microM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 microM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50 = 1.2 and 2.1 mM for aluminum lactate and AlCl3, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo

In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/d1 neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of beta-amyloid in the brains of individuals with Alzheimer disease requires the proteolytic cleavage of a membrane-associated precursor protein. The proteases that may be involved in this process have not yet been identified. Cathepsins are normally intracellular proteolytic enzymes associated with lysosomes; however, when sections from Alzheimer brains were stained by antisera to cathepsin D and cathepsin B, high levels of immunoreactivity were also detected in senile plaques. Extracellular sites of cathepsin immunoreactivity were not seen in control brains from age-matched individuals without neurologic disease or from patients with Huntington disease or Parkinson disease. In situ enzyme histochemistry of cathepsin D and cathepsin B on sections of neocortex using synthetic peptides and protein substrates showed that senile plaques contained the highest levels of enzymatically active cathepsin. At the ultrastructural level, cathepsin immunoreactivity in senile plaques was localized principally to lysosomal dense bodies and lipofuscin granules, which were extracellular. Similar structures were abundant in degenerating neurons of Alzheimer neocortex, and cathepsin-laden neuronal perikarya in various stages of disintegration could be seen within some senile plaques. The high levels of enzymatically competent lysosomal proteases abnormally localized in senile plaques represent evidence for candidate enzymes that may mediate the proteolytic formation of amyloid. We propose that amyloid precursor protein within senile plaques is processed by lysosomal proteases principally derived from degenerating neurons. Escape of cathepsins from the stringently regulated intracellular milieu provides a basis for an abnormal sequence of proteolytic cleavages of accumulating amyloid precursor protein

To investigate the role of proteolysis in amyloid formation, we studied the localization of the proteolytic enzymes, cathepsin D and cathepsin B, in the prefrontal cerebral cortex and hippocampus of human postmortem brains from patients with Alzheimer's disease and from individuals free of neurological disease. In control and Alzheimer brains, cathepsin immunoreactivity within cells was localized to lysosome-related structures, which were particularly abundant in neuronal perikarya. In Alzheimer brain, cathepsin immunoreactivity was also heavily concentrated extracellularly within senile plaques. Cathepsin immunoreactivity associated with plaques was not confined to lysosomes and was distributed throughout the plaque. Isolated amyloid cores, however, were not immunostained. Cathepsin-laden perikarya of degenerating neurons were frequently seen within senile plaques and, in the more advanced stages of degeneration, cathepsin immunoreactivity was present throughout the cytoplasm. Other identified constituents of senile plaques appeared to be less significant sources of cathepsin immunoreactivity, including astrocytes, degenerating neurites, microglia and macrophages. These results demonstrate that lysosomal proteinases are major constituents of the senile plaque and that degenerating neuronal perikarya are a principal source of the cathepsin immunoreactivity. We propose that the unregulated action of extracellular cathepsins liberated from degenerating neurons may lead to abnormal processing of the amyloid precursor protein and to the formation of amyloid locally within senile plaques in Alzheimer's disease

Antinuclear antibodies (ANA), antiphospholipid antibodies (APA), rheumatoid factor (RF), and immunoglobulin (Ig) M levels were determined in 184 male chronic psychiatric patients on long-term therapy with neuroleptics, and in 35 age-matched normal male controls. The prevalence of one or more of these autoantibodies was 70% in the neuroleptic-treated patients and 9% in the normal controls. Polyclonal IgM elevation was frequently seen among patients treated with phenothiazines. There was a significant correlation between the presence of ANA, APA, and RF; ANA and APA were more frequently associated with CPZ therapy, but the prevalence of RF was high in all treatment groups. These findings suggest that antibodies against the Fc fragment of IgG are the most common autoantibody associated with neuroleptics. Alternatively, the presence of RF could be a common finding in patients with neuropsychiatric disorders

To begin to understand the regulation and roles of neurofilament phosphorylation, we localized the phosphorylated domains on the 140-145-kDa neurofilament subunit (NF-M) and identified the protein kinases that may specifically phosphorylate the sites within these domains in vivo. Mouse retinal ganglion cells were labeled in vivo by injecting mice intravitreally with [32P]orthophosphate, and neurofilament-enriched fractions were obtained from the optic axons. Two-dimensional phosphopeptide map analysis of NF-M after digestion with alpha-chymotrypsin and trypsin revealed seven major (M8-M14) and at least eight minor (M1-M7 and M15) phosphopeptides. Two-dimensional phosphopeptide map analyses of NF-M phosphorylated in vitro by individual purified or endogenous axonal cytoskeleton-associated protein kinases showed that five peptides (M9-M13) were substrates for the heparin-sensitive second messenger-independent protein kinase(s). Protein kinase A and/or protein kinase C phosphorylated eight other peptides (M1-M8). Two alpha-chymotryptic peptides (C1 and C2) that were phosphorylated by protein kinase A but not by the endogenous independent kinase(s) were isolated by high performance liquid chromatography on a reverse-phase C8 column. Partial sequence analysis of peptides C1 (S R V S G P S ...) and C2 (S R G S P S T V S ...) showed that the peptides were localized on the head domain of NF-M at 25 and 41 residues from the amino terminus, respectively. Tryptic digest of peptide C1 (less than 12 kDa) generated the phosphopeptides M1-M6. Peptide C2 was a breakdown product of peptide C1. Since the polypeptide sites targeted by second messenger-independent kinase(s) associated with neurofilaments are localized on the carboxyl-terminal domain, separate aspects of NF-M function appear to be regulated by separate kinase systems that selectively phosphorylate head or tail domains of the polypeptide

Western blot analyses of total assembled microtubule fractions from NB2a/d1 neuroblastoma cells demonstrated that these cells are capable of post-translationally modifying alpha-tubulin by acetylation and detyrosination. Immunocytochemical analyses of NB2a/d1 cells differentiated with dbcAMP which had been processed under microtubule-stabilizing conditions demonstrated that all forms of alpha-tubulin were present throughout perikarya and neurites. By contrast, extraction of cells with Triton X-100 revealed a regional concentration of acetylated and detyrosinated alpha-tubulin subunits within axonal neurites, detectable in some cells after 3 days of differentiation and in nearly all cells after 7 days. Resistance of neurites to retraction following colchicine-treatment developed at a similar rate; furthermore, colchicine-resistant neurites contained intense acetylated alpha-tubulin immunoreactivity. We conclude that NB2a/d1 cells are capable of acetylating and detyrosinating alpha-tubulin subunits and that selective post-translational modification of alpha-tubulin subunits may be related to neuritic maturation

Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton

We used immunoblot and immunocytochemical methodologies to characterize the appearance and intracellular localization of the high molecular weight neurofilament subunit (NF-H) within the Triton-insoluble cytoskeleton during the first 5 days of differentiation of mouse NB2a/d1 neuroblastoma cells. Hypophosphorylated and partially phosphorylated forms of NF-H were detected in cells before and throughout differentiation. By contrast, some extensively phosphorylated forms of NF-H were first detected on the third day of differentiation and at least one additional 200 kDa isoform was visualized in cytoskeletons only after five days of differentiation. Extensively phosphorylated forms of NF-H were restricted to axonal neurites; by contrast, hypophosphorylated and partially phosphorylated forms of NF-H were present throughout undifferentiated and differentiated cells

A total of 27 subjects began active treatment in this double-blind study comparing the efficacy and safety of trazodone and fluoxetine in geriatric depressed patients, but only 13 completed 6 weeks on study medication. Both agents were effective according to weekly and endpoint analyses, and there was no evidence of significant effects on blood pressure, pulse, or weight. Separate analysis of patients who had received an adequate trial of medication indicated a trend toward relatively more fluoxetine-treated patients meeting clinical criteria for resolved depression